The invention relates to ionization with low fragmentation and adduct formation, of high-molecular weight analyte molecules, particularly of large biopolymers, by matrix-assisted laser desorption (MALDI) from liquid matrices for mass-spectrometric analysis of the resulting ions, particularly for determination of their molecular weight.
For mass-spectrometric determination of the molecular weight of biomolecules or other high polymers, ionization by means of matrix-assisted desorption with the aid of a pulsed UV laser (MALDI) has been established as a standard method in spite of some disadvantages. MALDI is also used for structural determination, generally then with desirable fragmentation of the molecule ions by taking special measures.
Normally, lasers are used which operate within the ultraviolet light range and emit light pulses lasting a few nanoseconds, such as simple and inexpensive nitrogen lasers with 337 nanometer wavelength and two to three nanoseconds light pulse length. The pulsed generation of ions calls for the use of time-of-flight mass spectrometers (TOF-MS) for analysis, although ion storage mass spectrometers such as ion cyclotron resonance mass spectrometers (FT-ICR=Fourier-transformation ion cyclotron resonance) or high frequency quadrupole ion trap mass spectrometers can also be used successfully.
Biomolecules in this context are the oligonucleotides (i.e. the genetic material in its various forms such as DNA or RNA), proteins and polysaccharides (i.e. the essential building blocks of the living world), including their particular analogs and conjugates, such as glycoproteins or lipoproteins. Other high polymers in particular are the artificially produced polymers. In the following, these biopolymers and artificial polymers, the molecules of which are to be analyzed, are simply called the xe2x80x9canalyte.xe2x80x9d
The selection of matrix substance for MALDI depends on the type of analyte molecules; in the meantime, several hundred various matrix substances have become known which are each suitable for specific groups of analyte molecules. The matrix substance in particular has the following tasks: 1) to bond the sample substance in a finely distributed, very dilute form to the sample support, 2) to absorptively collect the energy from the laser beam, 3) to blow the analyte molecules individually into the gas phase by creating a vapor cloud, and 4) to ionize a portion of the analyte molecules by means of protonation or deprotonation. During this process of vaporization and ionization, neither should the analyte molecules decompose, nor should matrix or other molecules attach themselves to the analyte ions to a larger extent, since determination of the correct molecular weight is no longer possible in either case.
It has generally proven favorable to use aromatic acids as matrix substances that are crystalline in a normal state, and to integrate relatively few analyte molecules into the small matrix crystals or at least imbed them in the boundary surfaces between the small crystals. The bio-molecules are generally at least weakly water soluble so it is preferable, though not necessary, to use water soluble matrix substances. A rule of thumb has been developed which states that the selection of matrix substances becomes more difficult, the higher the molecular weight of the biosubstances is. Either fragmentation of the macromolecules increases to such an extreme degree that molecule ions can no longer be found, or adducts with matrix or other molecules are formed so that it is hardly possible to determine the molecular weight. In many cases, a mixture of adduct formation and splitting off of smaller fragments causes the mass-spectrometric signal to become a broad peak, which makes precise mass determination no longer possible.
In constantly searching for new matrix substances more favorable for certain substance categories, it has almost been forgotten that the first MALDI ionization of high-molecular substances was observed in a liquid glycerol matrix, to which a fine metallic powder was added for the absorption of UV laser beams (K. Tanaka et al., Rapid Comm. in Mass Spectrom., 2, 151, 1988). As MALDI was further developed, glycerol was used only occasionally as the matrix due to the disadvantages described below, but also in particular because glycerol cannot be excited directly by the standard UV lasers. In principle, however, this disadvantage was eliminated by a working group dissolving UV absorbents within the glycerol and thus adapting the liquid mixed matrix to the standard UV lasers, although, for unknown reasons, not all the absorbents displayed good results, and some strong UV absorbents actually prevented ionization (D. S. Cornett et al., xe2x80x9cLiquid Mixtures for Matrix-Assisted Laser Desorptionxe2x80x9d, Anal. Chem., 65, 2608, 1993).
In a very recent article, an infrared laser was used for ionization, the radiation of which was capable of directly exciting one of the stretching vibrations of the glycerol (xe2x80x9cInfrared MALDI Mass Spectrometry of Large Nucleic Acidsxe2x80x9d, Science 281, 2212, 1998). In particular, the radiation from an erbium-YAG laser with 2.94 micrometers wavelength excites the stretching vibrations of the OH groups. It was established that this combination of glycerol and infrared radiation with extremely low-energy photons is capable of extraordinarily sensitive, low-fragment ionization of molecules with extremely high molecular weights. Although the erbium-YAG infrared laser, most favorable for absorption of radiation, is indeed still relatively expensive and technically not yet particularly reliable, it can be expected that this type of laser will go through development similar to efficient equipment like the related neodym-YAG laser.
Glycerol is at all suitable for this type of ionization because it has a relatively low vapor pressure. Even in a vacuum, a small droplet of about one microliter evaporates quite slowly, taking about one half hour to dry out completely. This time can be utilized for MALDI analysis of several samples on a carrier plate.
Glycerol was used already one or two decades ago as a liquid medium for the ionization of dissolved substances by means of bombardment with fast neutral particles (fast atom bombardment, xe2x80x9cFABxe2x80x9d). Using this method, highly sensitive, very low fragment and low adduct spectra of molecules with relatively high molecular weights were obtained.
So far there can only be speculation about the reason for the similarly high ionization effect of glycerol in these very diverse ionization methods. It appears possible that macromolecules which are almost always composed of mixed hydrophobic and hydrophilic groups (amphiphitic substances) prefer to keep their more hydrophobic side toward the surface, and project their hydrophilic side toward the very polar solution. This effect may lead to a substantial increase in the concentration of these high polymers at the surface. On the other hand, glycerol (1,2,3-propantriol) as a trihydroxylic alcohol may possibly ionize other substances very easily by means of proton donation from one of the alcohol groups. Even shockwaves propagated in the liquid with a shaking off of surface molecules has been discussed. It is also known that very polar water may be used as a matrix, but it requires extreme cooling of the carrier plates within a vacuum to prevent immediate evaporation.
Ionization by means of glycerol offers advantages, but also severe disadvantages.
Advantages: In addition to the high sensitivity for very large molecules and the relatively low fragmentation and adduct-formation, the uniform ionization yield over the entire drop surface is especially prominent on the list of advantages. Since visual control of the bombardment site is no longer necessary, and automated procedure is possible, different than the case previously for MALDI procedures where droplets are dried into solid matrices. Also considered advantageous is the fact that samples are relatively easy to prepare; a droplet with aqueous analyte solution may simply be applied to a droplet of glycerol. The preparation is conducted very simply in pipetting machines. The water evaporates (to a large extent at least) when introduced into the vacuum of the mass spectrometer.
Disadvantages: Prominent on the list of disadvantages are the relatively short time available for use and the extreme load on the vacuum system; due to both this vacuum load as well as its short time available for use, hundreds of samples unfortunately cannot be analyzed on a sample support using this method. A detectable load on the vacuum system, and thus also on the quality of the spectra, occurs with only ten samples on a support, and the quality is affected by the poor pressure within the spectrometer. This runs counter to the increasing demand for a high sample throughput where not just ten, but rather a thousand samples are required on a sample support. This disadvantage can be offset by extreme cooling of the sample support plate outside of the vacuum, in the sample support lock, and within the vacuum, although such cooling is difficult (the carrier plate is at a potential of 30 kV in the mass spectrometer) and not available in commercial mass spectrometers.
Of course, for this high analysis sample throughput, it is essential not only for the MALDI ionization to be automatable, but also for all the analysis steps including preparation of the samples. However, as already mentioned above, this is exactly the case when glycerol is used as the matrix substance, and it is generally better than when solid matrix substances are used.
The invention consists of using liquid matrices made up of multihydroxylic (at least trihydroxylic) alcohols for MALDI, which however possess a substantially lower vapor pressure compared to the glycerol presently used by extension of the carbon chains and integration of at least one ether bond. Diglycerin, Triglycerin, and Polyglycerin (trivial names, supplier: Solvay Alkali GmbH, Dilsseldorf; in the following designated as diglycerol, triglycerol, and polyglycerol) belong to this group of liquids. Here, the matrix molecules may be directly excited by use of infrared lasers, or adapted to other types of lasers by resolving absorbents for light at other wavelengths.
It has been shown in experiments that a large portion of OH groups (hydroxy groups) is vital for the functioning of a MALDI matrix substance. On the one hand (in addition to the chain extension itself), the large share of these alcoholic OH groups contributes to reduction of the vapor pressure, but it also appears to be important for energy consumption, for concentration of analyte molecules at the surface and for ionization. In the case of erbium-YAG lasers, these hydroxy groups are also particularly helpful in the direct consumption of energy from the laser radiation.
Tetrahydroxylic alcohols of normal hydrocarbons (the simplest sugars) are already solid, thus the basic idea of the invention of using liquids rich in hydroxy groups with extremely low vapor pressure cannot be realized with normal hydrocarbon alcohols. The integration of ethereal bonds in the carbon chains do however keep higher alcohols liquid, so that matrix liquids of the type according to the invention can be verified by means of ether polyols, or by polyether polyols.
Belonging particularly to these compounds are tetravalent diglycerol (trivial name) with the structure HOH2Cxe2x80x94HOHCxe2x80x94H2Cxe2x80x94Oxe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2OH or the pentavalent triglycerol HOH2Cxe2x80x94HOHCxe2x80x94H2Cxe2x80x94Oxe2x80x94CH2xe2x80x94CHOHxe2x80x94H2Cxe2x80x94Oxe2x80x94CH2xe2x80x94CHOHxe2x80x94CH2OH, which has two ether bonds. The structure resembles so-called polyethylene glycols, which are used as ultra high vacuum pump oils due to their extremely low vapor pressure, although in spite of relatively long chain lengths, they represent only dihydroxylic, terminal alcohols and have proven in experiments to be unsuitable for the present purpose.
Diglycerol, triglycerol and higher polyglycerols, including those of an asymmetrical type (such as glycol glycerol joined by an ether bond), are all referred to here under the term polyglycerols for purposes of simplification.
The polyglycerols are miscible with water and can dissolve practically all analyte molecules if only introduced in sufficiently low concentrations. The solubility for many substances, such as for some favorable UV absorbents, is greatly reduced however with a higher degree of polymerisation. Due to its miscibility with water, a method can be used in which the polyglycerols are first applied to the carrier plates, onto which the aqueous analyte solutions are then simply pipetted. The polyglycerols have a thick, oily consistency, similar to honey at a higher degree of polymerisation. However, different than oils, they very easily wet hydrophilic surfaces due to their strong polarity; they can therefore be applied to MALDI carrier plates simply by using multi-needle heads.
The vapor pressure is already so low for diglycerol that either 96 or 384 samples can be applied, for example, without substantially disturbing the mass spectrometer vacuum. Even the application of 1,536 small-area samples is possible. These numbers correspond to the numbers of reaction containers in the standard micro-titration plates for parallel preparation of samples in biochemistry.
It has also proven advantageous if the carrier plates are already provided with a hydrophobic basic pattern that has hydrophilic anchorage areas for the polyglycerols. The anchorage areas should be large enough that the polyglycerols form very flat droplets. Droplets of about one millimeter diameter and about 0.2 to 0.4 millimeters height are advantageous, for example. Such a basic pattern on the carrier plate with hydrophobic marginal areas for the droplets prevents the pipetted water droplets with analyte molecules from polyglycerols from flowing over to adjacent areas of the carrier plate before they can be detached. The sample spots, and thus also the evaporation rate of the polyglycerols, stay very small. On a 78 by 114 millimeter carrier plate (size of micro-titration plates), 1,536 sample spots can thus be easily applied.
Already for diglycerol, and even more so for triglycerol, the pressure within the mass spectrometer is barely influenced even if large numbers of small-area samples are applied to a sample support. For diglycerol, the droplets"" shrinkage is only barely perceptible even when kept for an entire day within the vacuum. Thus analysis times of at least a day can be realized. This allows analysis times of about one minute for each sample with 1,536 samples, much more than necessary.
The water from the aqueous solution of analyte molecules is conveniently evaporated out of the sample spots for practical reasons before introducing the carrier plates into the vacuum of the mass spectrometer""s ion source. This evaporation of water should be conducted carefully since remnants of water occasionally can lead to explosion of the droplets during laser bombardment. The evaporation may occur under reduced pressure and should be supported by careful heating of the carrier plate to temperatures of 80 to 130xc2x0 C. (depending on the type of analyte molecules). In this way, evaporation can proceed in the vacuum lock of the mass spectrometer although a special vacuum chamber is more favorable for this purpose due to the extended period of the evaporation process.
Liquid matrices according to the invention, with extremely low vapor pressure, can also be exploited advantageously in entirely different applications. Thus it is possible to apply these matrix liquids to blot membranes, which are charged in a known manner by blotting with electrophoretically, two-dimensionally separated proteins from PAGE plates (PAGE=polyacryl gel electrophoresis). The protein molecules adsorbed on the porous surface structures of the blot membranes are partially detached by the matrix liquids and applied to their surface by difflusion. Here, by controlling the viscosity via temperature and water content, it is possible to disturb the special separation of the proteins as little as possible. A capacity for storage can be achieved by evaporation of the water and freezing of the matrix liquid. Highly sensitive analysis proceeds by means of MALDI ionization directly from the blot, membrane stretched onto a carrier plate, or by simple transfer (imprinting, stamping) of the matrix liquid onto a special carrier plate and, by means of two-dimensional array analysis, by corresponding movement of the carrier plate.